Fatty acid hydroxylases and uses thereof

ABSTRACT

The invention provides isolated nucleic acid molecules which encode novel fatty acid hydroxylases. The invention also provides recombinant expression vectors including hydroxylase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for the production of hydroxyl fatty acids such as 12-hydroxyoctadec-9-enoic acid (ricinoleic acid).

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/815,774, filed Jun. 22, 2006, the entire contents of which are herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

Fatty acids are carboxylic acids with long-chain hydrocarbon side groupsand play a fundamental role in many biological processes. Fatty acidsare often unhydroxylated; however, such unhydroxylated fatty acids maybe converted to hydroxyl fatty acids by the introduction of at least onehydroxyl group, a process catalyzed by a hydroxylase enzyme.

Hydroxyl fatty acids and hydroxyl oils are particularly important for avariety of industrial applications. Indeed, hydroxyl fatty acids, suchas ricinoleic acid (12-hydroxyoctadec-9-enoic acid), are importantindustrial feedstock in the manufacture of biolubricants, functionalfluids, ink, paints, coatings, nylons, resins, foams and otherbiopolymers.

The biosynthesis of fatty acids is a major activity of plants andmicroorganisms. Biotechnology has long been considered an efficient wayto manipulate the process of producing fatty acids in plants andmicroorganisms. It is cost-effective and renewable with little sideeffects. Thus, tremendous industrial effort directed to the productionof various compounds including specialty fatty acids and pharmaceuticalpolypeptides through the manipulation of plant, animal, andmicroorganismal cells has ensued.

At present, however, castor bean (Ricinus communis) is the onlycommercial source for hydroxyl fatty acids. Due to poor agronomicperformance and the presence of highly potent toxins (ricin) andallergens in the seed, castor bean is not an ideal source for the fattyacids. Thus, a growing demand exists for alternatives to replace castorbean as a source of the hydroxyl fatty acids (Jaworski and Cahoon,2003). Genes involved in the biosynthesis of hydroxyl fatty acids suchas ricinoleic and lesqueroleic acids have been isolated from plantcastor bean (Ricinus communis) and Lesquerella fendleri (van de Loo etal., 1995) (Broun et al., 1998). Both genes encode oleate12-hydroxylase, which introduces a hydroxyl group at position 12 ofoleic acid. However, the introduction of the castor bean oleatehydroxylase into tobacco, Arabidopsis thaliana resulted in low tointermediate levels of ricinoleic acid accumulation in seeds (van de Looet al., 1995) (Broun and Somerville, 1997) (Smith et al., 2003).

Although biotechnology offers an attractive route for the production ofspecialty fatty acids, current techniques fail to provide an efficientmeans for the large scale production of hydroxyl fatty acids.Accordingly, there exists a need for an improved and efficient method ofproducing hydroxyl fatty acids, such as ricinoleic acid.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery of anucleic acid molecule encoding a novel fatty acid hydroxylase fromClaviceps purpurea. In particular, the fatty acid hydroxylase of theinvention is capable of catalyzing the introduction of a hydroxyl group,for example, at position 12 of a fatty acid such as oleic acid. In aparticular embodiment, the fatty acid hydroxylase is capable ofcatalyzing the introduction of a hydroxyl group at position 12 ofoctadec-9-enoic acid to form 12-hydroxyoctadec-9-enoic acid (ricinoleicacid). For example, the expression of the Claviceps purpurea fatty acidhydroxylase (CpFAH) in Saccharomyces cerevisiae resulted in theproduction of 12-hydroxyoctadec-9-enoic acid through the introduction ofa hydroxyl group at position 12 of octadec-9-enoic acid.

The use of the nucleic acid molecules and polypeptides of the presentinvention provides a means for modulating, for example, enhancing, theproduction of desired hydroxyl fatty acids. For example, theintroduction of these hydroxylase nucleic acid and polypeptide moleculesin microbial and plant cells, such as Brassica juncea, for example,under the control of a seed-specific promoter, will allow for theenhanced production of hydroxyl fatty acids such as ricinoleic acid.

In one aspect, the present invention is directed to an isolated nucleicacid molecule selected from the group consisting of a) an isolatednucleic acid molecule encoding a fatty acid hydroxylase from the genusClaviceps, or a complement thereof; b) an isolated nucleic acid moleculeincluding the nucleotide sequence of SEQ ID NO:1, or a complementthereof; c) an isolated nucleic acid molecule which encodes apolypeptide including the amino acid sequence of SEQ ID NO:2, or acomplement thereof; d) an isolated nucleic acid molecule which encodes anaturally occurring allelic variant of a polypeptide including the aminoacid sequence of SEQ ID NO:2, or a complement thereof; e) an isolatednucleic acid molecule including a nucleotide sequence which is at least70% identical to the entire nucleotide sequence of SEQ ID NO:1, or acomplement thereof; f) an isolated nucleic acid molecule including anucleotide sequence which hybridizes to the complement of the nucleotidesequence of SEQ ID NO: 1 under stringent conditions, or a complementthereof; and g) an isolated nucleic acid molecule including a fragmentof at least 15 contiguous nucleotides of the entire nucleotide sequenceof SEQ ID NO:1, or a complement thereof. In a particular embodiment, thenucleic acid molecule encodes a fatty acid hydroxylase protein having anactivity of catalyzing the introduction of a hydroxyl group in a fattyacid. Alternatively or in addition, the nucleic acid molecule encodes aprotein having desaturase activity, for example, Δ12 desaturaseactivity. In another embodiment, the isolated nucleic acid moleculefurther includes a nucleotide sequence encoding a heterologouspolypeptide.

In another aspect, the invention is directed to a vector, for example,an expression vector, including a nucleic acid molecule of theinvention. In a particular embodiment, the nucleic acid molecule may beunder the control of a seed-specific promoter, for example, Conlinin 1,Conlinin 2, napin and LuFad3.

In another aspect, the invention is directed to a host cell transfectedwith the expression vector including a nucleic acid molecule of theinvention. The host cell may be a plant cell, for example, a plant cellfrom an oilseed crop, including, but not limited to, flax (Linum sp.),rapeseed (Brassica sp.), soybean (Glycine and Soja sp.), sunflower(Helianthus sp.), cotton (Gossypium sp.), corn (Zea mays), olive (Oleasp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), peanut(Arachis sp.), hemp, camelina, crambe, oil palm, coconuts, groundnuts,sesame seed, castor bean, lesquerella, tallow tree, sheanuts, tungnuts,kapok fruit, poppy seed, jojoba seeds and perilla. Alternatively, thehost cell may be a microbial cell, including, but not limited to,Candida, Cryptococcus, Lipomyces, Rhodosporidium, Yarrowia,Thraustochytrium, Pythium, Schizochytrium and Crythecodinium.

In another aspect, the invention provides a method of producing apolypeptide by culturing a host cell of the invention in an appropriateculture medium to, thereby, produce the polypeptide, for example, afatty acid hydroxylase.

In yet another aspect, the invention provides isolated polypeptidesselected from the group consisting of a) an isolated fatty acidhydroxylase polypeptide from Claviceps; b) an isolated polypeptideincluding the amino acid sequence of SEQ ID NO:2; c) an isolatedpolypeptide including a naturally occurring allelic variant of apolypeptide including the amino acid sequence of SEQ ID NO:2; d) anisolated polypeptide including an amino acid sequence encoded by anucleic acid molecule including the nucleotide sequence of SEQ ID NO: 1;e) an isolated polypeptide which is encoded by a nucleic acid moleculeincluding a nucleotide sequence which is at least 70% identical to theentire nucleotide sequence of SEQ ID NO:1; f) an isolated polypeptideincluding an amino acid sequence which is at least 70% identical to theentire amino acid sequence of SEQ ID NO:2; and g) an isolatedpolypeptide including a fragment of a polypeptide including the aminoacid sequence of SEQ ID NO:2, wherein the polypeptide fragment maintainsa biological activity of the complete polypeptide. In a particularembodiment, the polypeptide is involved in the production of a hydroxylfatty acid. Alternatively or in addition, the polypeptide has adesaturase activity, for example, a Δ12 desaturase activity. In anotherembodiment, the polypeptide also includes a heterologous amino acidsequence.

In another aspect, the invention provides a method for producing ahydroxyl fatty acid by culturing a host cell of the invention such thatthe hydroxyl fatty acid is produced. In another embodiment, theinvention provides a method for producing a hydroxyl fatty acid bycontacting a composition including at least one hydroxylase targetmolecule with at least one polypeptide of the invention under conditionssuch that the hydroxyl fatty acid is produced. In yet another aspect,the invention provides a method of producing a cell capable ofgenerating a hydroxyl fatty acid by introducing into the cell a nucleicacid molecule of the invention, wherein the nucleic acid moleculeencodes a hydroxylase having an activity of catalyzing the introductionof a hydroxyl group in a fatty acid. In yet another aspect, the presentinvention is directed to a method of modulating, for example, enhancing,the production of a hydroxyl fatty acid by culturing a cell transformedwith the expression vector of the invention, such that modulation of theproduction of a hydroxyl fatty acid occurs. In a further aspect, thepresent invention is directed to a method for the large scale productionof a hydroxyl fatty acid by culturing a cell transformed with theexpression vector of the invention. In certain embodiments, theexpression of the nucleic acid molecule results in the modulation of theproduction of the hydroxyl fatty acid, 12-hydroxyoctadec-9-enoic acid(ricinoleic acid). Additionally, the hydroxylase target molecule or theunhydroxylated fatty acid may be octadec-9-enoic acid (oleic acid).

In one embodiment, the hydroxyl fatty acid produced by the foregoingmethods may be recovered from the culture. In another embodiment thecell is a plant cell, for example, an oilseed plant, including, but notlimited to, flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycineand Soja sp.), sunflower (Helianthus sp.), cotton (Gossypium sp.), corn(Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa(Theobroma cacoa), peanut (Arachis sp.), hemp, camelina, crambe, oilpalm, coconuts, groundnuts, sesame seed, castor bean, lesquerella,tallow tree, sheanuts, tungnuts, kapok fruit, poppy seed, jojoba seedsand perilla. In a particular embodiment, the cell belongs to the genusArabidopsis. In another embodiment, the cell is Brassica juncea. In yetanother embodiment, the cell is a microbial cell, for example, Candida,Cryptococcus, Lipomyces, Rhodosporidium, Yarrowia, Thraustochytrium,Pythium, Schizochytrium and Cythecodinium.

In yet another aspect, the present invention is directed to a host cellhaving a) a nucleic acid molecule including the nucleotide sequence ofSEQ ID NO:1, wherein the nucleic acid molecule is disrupted by at leastone technique selected from the group consisting of a point mutation, atruncation, an inversion, a deletion, an addition, a substitution andhomologous recombination, for example, such that the fatty acidhydroxylase activity and/or desaturase activity is disrupted; b) anucleic acid molecule having the nucleotide sequence of SEQ ID NO:1,wherein the nucleic acid molecule includes one or more nucleic acidmodifications as compared to the sequence set forth in SEQ ID NO:1,wherein the modification is selected from the group consisting of apoint mutation, a truncation, an inversion, a deletion, an addition anda substitution, for example, such that the modified nucleic acidmolecule encodes for a polypeptide retaining fatty acid hydroxylaseand/or desaturase activity; or c) a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1, wherein the regulatory region of thenucleic acid molecule is modified relative to the wild-type regulatoryregion of the molecule by at least one technique selected from the groupconsisting of a point mutation, a truncation, an inversion, a deletion,an addition, a substitution and homologous recombination, for example,so as to modify, for example, enhance fatty acid hydroxylase and/ordesaturase activity.

In other aspects, the invention is directed to a plant including avector described herein, and oils or seeds produced by the plant. Inanother aspect, the invention is directed to a composition including theoil and/or seed, wherein the composition comprises a product selectedfrom the group consisting of a biolubricant, a functional fluid, an ink,a paint, a coating, a nylon, a resin, a foam and a biopolymer. Inanother aspect, the invention is directed to a hydroxyl fatty acidobtained by a method described herein. In a further aspect, theinvention is directed to compositions including the hydroxyl fatty acidsproduced by a method described herein, wherein the composition is aproduct selected from the group consisting of a biolubricant, afunctional fluid, an ink, a paint, a coating, a nylon, a resin, a foamand a biopolymer.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and amino acid sequence of an oleatehydroxylase from Claviceps purpurea (CpFAH) as follows: (A) the cDNAsequence of the open reading frame (SEQ ID NO:1); (B) the translatedprotein sequence (SEQ ID NO:2) and (C) the cDNA aligned with thetranslated amino acid sequence.

FIG. 2 shows an alignment of the amino acid of oleate hydroxylase fromClaviceps purpurea (SEQ ID NO: 2) versus that of other fatty acidhydroxylases and related enzymes including those from A. nidulans(AnOdeA) (SEQ ID NO: 3), Lesquerella fendleri (LfFAH) (SEQ ID NO: 4),and Ricinus communis (RcFAH) (SEQ ID NO: 5).

FIG. 3B is a gas chromatographic (GC) analysis of the expression offatty acids in an experimental strain of yeast transformed with CpFAH ascompared to a control strain of yeast (FIG. 3A). The peak labeled12OH-18:1-9 represents the presence of a fatty acid unique to the yeaststrain transformed with CpFAH.

FIG. 4B is a gas chromatographic/mass spectroscopy (GC/MS) analysis ofthe peak unique to the experimental strain of yeast as depicted in FIG.3B (i.e., 12OH-18:1-9) as compared to the GC/MS analysis of StandardTMS-methylricinoleate (FIG. 4A).

FIG. 5 is a depiction of an expression vector including CpFAH designedfor large scale production of hydroxyl fatty acids in plants.

FIG. 6A is a gas chromatographic (GC) analysis of TMS-derivatized fattyacid methyl esters prepared from a single seed of Arabidopsis thalianadouble mutant (fad2fae1) transformed with CpFAH as compared to a controluntransformed strain of fad2fae1 (FIG. 6B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel fatty acid hydroxylase family members, referred to interchangeablyherein as “hydroxylases” or “hydroxylase” nucleic acid and proteinmolecules. These novel molecules are members of the fatty acidhydroxylase family and are expressed in the hydroxyl fattyacid-producing organisms Claviceps purpurea (C. purpurea). The presentinvention is further based, at least in part, on the discovery that theC. purpurea fatty acid hydroxylase (CpFAH) of the invention, andsufficiently homologous hydroxylases thereof, catalyze the introductionof a hydroxyl group in a fatty acid. The present invention is furtherbased, at least in part, on the discovery that the C. purpurea fattyacid hydroxylase, and sufficiently homologous hydroxylases thereof,catalyze the introduction of a double bond, for example at position 12of a fatty acid, such as oleic acid.

As used herein, the term “fatty acids” is art recognized and includes along-chain hydrocarbon based carboxylic acid. Fatty acids are componentsof many lipids including glycerides. The most common naturally occurringfatty acids are monocarboxylic acids which have an even number of carbonatoms (16 or 18). Fatty acids may be hydroxylated or unhydroxylated.Hydroxylated fatty acids contain a hydroxyl group at least one positionalong the fatty acid chain.

The controlling steps in the production of hydroxyl fatty acids, i.e.,the hydroxyl fatty acid biosynthetic pathway, are catalyzed by fattyacid hydroxylases, e.g., C. purpurea fatty acid hydroxylases (CpFAH).Specifically, such enzymes catalyze the formation of a hydroxyl group ona carbon atom of a fatty acid molecule. As used herein, the term“hydroxyl fatty acid biosynthetic pathway” refers to a series ofchemical reactions leading to the synthesis of a hydroxyl fatty acideither in vivo or in vitro. Fatty acid hydroxylases such as CpFAHintroduce a hydroxyl group into oleic acid (18:1-9) resulting information of ricinoleoc acid (12-OH-18:1-9).

The term “family” when referring to the protein and nucleic acidmolecules of the present invention is intended to mean two or moreproteins or nucleic acid molecules having a common structural domain ormotif, for example, at least one of the conserved amino acid domains,GHECGH (SEQ ID NO: 6), HSAHH (SEQ ID NO: 7) and HVVHH (SEQ ID NO: 8),and having sufficient amino acid or nucleotide sequence homology asdefined herein. Such family members can be naturally or non-naturallyoccurring and can be from either the same or different species. Forexample, a family can contain a first protein of human origin as well asother distinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics. For example,the family of hydroxylase proteins of the present invention are involvedin the introduction of a hydroxyl group, for example, in a fatty acid.

Isolated hydroxylase proteins of the present invention have an aminoacid sequence sufficiently homologous to the amino acid sequence of SEQID NO:2 or are encoded by a nucleotide sequence sufficiently homologousto SEQ ID NO:1. As used herein, the term “sufficiently homologous”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., an aminoacid residue which has a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences share commonstructural domains or motifs, for example, those domains or motifsconserved among the various fatty acid hydroxylases depicted in FIG. 2,and/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more homology or identity across the domains and contain at leastone and preferably two structural domains or motifs, are defined hereinas sufficiently homologous. Furthermore, amino acid or nucleotidesequences which share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or more homology or identity and share acommon functional activity are defined herein as sufficientlyhomologous.

As used interchangeably herein, a “hydroxylase activity,” “biologicalactivity of a hydroxylase,” or “functional activity of a hydroxylase,”includes an activity exerted or mediated by a hydroxylase protein,polypeptide or nucleic acid molecule on a hydroxylase responsive cell oron a hydroxylase substrate, as determined in vivo or in vitro, accordingto standard techniques. In one embodiment, a hydroxylase activity is adirect activity such as an association with a hydroxylase targetmolecule. As used herein, a “target molecule” or “binding partner” is amolecule, for example, a molecule involved in the synthesis of hydroxylfatty acids, e.g., an intermediate fatty acid (such as a hydroxylatedfatty acid on which the incorporation of further hydroxyl groups isdesired) or an unhydroxylated fatty acid, with which a hydroxylaseprotein binds or interacts in nature such that a hydroxylase-mediatedfunction is achieved. In a particular embodiment, the target molecule orbinding partner is octadec-9-enoic acid (oleic acid). A hydroxylasedirect activity also includes the formation of a hydroxyl group on afatty acid molecule to form a hydroxyl fatty acid molecule. For purposesof the present invention, the hydroxylase may introduce a hydroxyl groupto an entirely unhydroxylated fatty acid or, alternatively, mayintroduce an additional hydroxyl group to a previously hydroxylatedfatty acid.

The nucleotide sequence of the isolated Claviceps purpurea hydroxylase(CpFAH) cDNA and the predicted amino acid sequence encoded by the CpFAHcDNA are shown in FIG. 1. The Claviceps purpurea CpFAH gene (the openreading frame), which is approximately 1434 nucleotides in length,encodes a protein which is approximately 477 amino acid residues inlength. The present invention is based, at least in part, on thediscovery that the CpFAH molecule is a bifunctional enzyme with bothhydroxylase and Δ¹² desaturase activity. For example, the CpFAH moleculecatalyzes the introduction of a hydroxyl group and/or a double bond infatty acids, for example at position 12 of oleic acid.

As used herein, “oleic acid” refers to a monounsaturated omega-9 fattyacid found in various animal and vegetable sources. Oleic acid has theformula C₁₈H₃₄O₂ (or CH₃(CH₂)₇CH═CH(CH₂)₇COOH) and is also known ascis-9-octadecenoic acid, octadec-9-enoic acid, 18:1-9 and 18:1 cis-9.

As used herein, “ricinoleic acid” refers to an unsaturated omega-9 fattyacid. Ricinoleic acid has the formula C₁₈H₃₄O₃ and is also known as12-hydroxyoctadec-9-enoic acid.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode hydroxylase proteins or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify hydroxylase-encoding nucleic acidmolecules (e.g., hydroxylase mRNA) and fragments for use as PCR primersfor the amplification or mutation of hydroxylase nucleic acid molecules.As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated hydroxylase nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or a portion of thenucleic acid sequence of SEQ ID NO:1, as hybridization probes,hydroxylase nucleic acid molecules can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook, J.et al. Molecular Cloning: A Laboratory Manual. 2^(nd), ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, can be isolated by the polymerase chain reaction (PCR) usingsynthetic oligonucleotide primers designed based upon the sequence ofSEQ ID NO:1.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to hydroxylase nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In still another embodiment, an isolated nucleic acid molecule of theinvention includes the complement of the nucleotide sequence shown inSEQ ID NO: 1, or a portion thereof. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1 is onewhich is sufficiently complementary to the nucleotide sequence shown inSEQ ID NO:1, such that it can hybridize to the nucleotide sequence shownin SEQ ID NO:1, thereby forming a stable duplex. In a particularembodiment, the complementary sequences of the invention are exactcomplements of the nucleic acid molecules of the invention, for example,a nucleotide sequence of SEQ ID NO:1, a nucleotide sequence encoding apolypeptide of SEQ ID NO:2, or an allelic variant thereof, and anucleotide sequence of at least 70% identity to the nucleotide sequenceof SEQ ID NO:1. For example, the complement may be a full and completecomplement of a nucleic acid molecule of the invention, for example, thenucleotide sequence of SEQ ID NO:1.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleotide sequence which is at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at leastabout 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, morepreferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, 91%, 92%,93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%,99% or more identical to the nucleotide sequence of SEQ ID NO:1 (e.g.,to the entire nucleotide sequence of SEQ ID NO:1), or a portion or acomplement thereof. Ranges and identity values intermediate to theabove-recited ranges, (e.g., 70-90% identical or 80-95% identical) arealso intended to be encompassed by the present invention. For example,ranges of identity values using a combination of any of the above valuesrecited as upper and/or lower limits are intended to be included.

In one embodiment, a nucleic acid molecule of the present inventioncomprises a nucleotide sequence which is at least (or no greater than)50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 1000 or 1250 or more nucleotides in length and hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule of SEQ ID NO:1.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, for example, afragment which can be used as a probe or primer or a fragment encoding aportion of a hydroxylase protein, e.g., a biologically active portion ofa hydroxylase protein. The nucleotide sequence determined from thecloning of the hydroxylase gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other hydroxylasefamily members, as well as hydroxylase homologues from other species.The probe/primer (e.g., oligonucleotide) typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO:1, of ananti-sense sequence of SEQ ID NO: 1, or of a naturally occurring allelicvariant or mutant of SEQ ID NO: 1.

Exemplary probes or primers are at least (or no greater than) 12 or 15,20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides inlength and/or comprise consecutive nucleotides of an isolated nucleicacid molecule described herein. Also included within the scope of thepresent invention are probes or primers comprising contiguous orconsecutive nucleotides of an isolated nucleic acid molecule describedherein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 baseswithin the probe or primer sequence. Probes based on the hydroxylasenucleotide sequences can be used to detect (e.g., specifically detect)transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment, a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of ahydroxylase sequence, e.g., a domain, region, site or other sequencedescribed herein. The primers should be at least 5, 10, 15, 20, 25, 30,35, 40, 45 or 50 base pairs in length and less than 100, or less than200, base pairs in length. The primers should be identical, or differ byno greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases when compared to asequence disclosed herein or to the sequence of a naturally occurringvariant. Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress a hydroxylase protein, suchas by measuring a level of a hydroxylase-encoding nucleic acid in asample of cells from a subject, e.g., detecting hydroxylase mRNA levelsor determining whether a genomic hydroxylase gene has been mutated ordeleted.

A nucleic acid fragment encoding a “biologically active portion of ahydroxylase protein” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO: 1, which encodes a polypeptide havinga hydroxylase biological activity (the biological activities of thehydroxylase proteins are described herein), expressing the encodedportion of the hydroxylase protein (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of thehydroxylase protein using standard assay techniques known in the art orthose techniques described, for example, in the Examples set forthherein. In an exemplary embodiment, the nucleic acid molecule is atleast 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 1000 or 1250 or more nucleotides in length and encodes aprotein having a hydroxylase activity (as described herein).

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1 due to degeneracy ofthe genetic code and thus encode the same hydroxylase proteins as thoseencoded by the nucleotide sequence shown in SEQ ID NO:1. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequencewhich differs by at least 1, but no greater than 5, 10, 20, 50 or 100amino acid residues from the amino acid sequence shown in SEQ ID NO:2.In yet another embodiment, the nucleic acid molecule encodes the aminoacid sequence of human hydroxylase. If an alignment is needed for thiscomparison, the sequences should be aligned for maximum homology.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non-naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population) that lead to changes inthe amino acid sequences of the hydroxylase proteins. Such geneticpolymorphism in the hydroxylase genes may exist among individuals withina population due to natural allelic variation.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding ahydroxylase protein, e.g., oilseed hydroxylase protein, and can furtherinclude non-coding regulatory sequences, and introns.

Accordingly, in one embodiment, the invention features isolated nucleicacid molecules which encode a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:2. Moreover,the nucleic acid molecule may hybridize to a complement of a nucleicacid molecule comprising SEQ ID NO:1, for example, under stringenthybridization conditions.

In addition to the C. purpurea fatty acid hydroxylase of SEQ ID NO: 1,it will be appreciated by those of ordinary skill in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the hydroxylase proteins may exist within a population (e.g., the C.purpurea population). Such genetic polymorphism in the fatty acidhydroxylase gene may exist among individuals within a population due tonatural variation. Such natural variations can typically result in 1-5%variance in the nucleotide sequence of the HA gene. Allelic variants ofthe CpFAH hydroxylase include both functional and non-functionalhydroxylase proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the hydroxylase protein thatmaintain the ability to, e.g., (i) interact with a hydroxylase substrateor target molecule (e.g., a fatty acid); and/or (ii) form a hydroxylgroup in a hydroxylase substrate or target molecule. Functional allelicvariants will typically contain only a conservative substitution of oneor more amino acids of SEQ ID NO:2, or a substitution, deletion orinsertion of non-critical residues in non-critical regions of theprotein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the hydroxylase protein that do not have theability to, e.g., (i) interact with a hydroxylase substrate or targetmolecule (e.g., a fatty acid such as an unhydroxylated fatty acid);and/or (ii) form a hydroxyl group in a hydroxylase substrate or targetmolecule. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or prematuretruncation of the amino acid sequence of SEQ ID NO:2, or a substitution,insertion, or deletion in critical residues or critical regions of theprotein.

The present invention further provides orthologues (e.g., humanorthologues of the hydroxylase proteins). Orthologues of the C. purpureahydroxylase proteins are proteins that are isolated from other organismsand possess the same hydroxylase substrate or target molecule bindingmechanisms and/or hydroxyl group forming mechanisms. Orthologues of theC. purpurea hydroxylase proteins can readily be identified as comprisingan amino acid sequence that is substantially homologous to SEQ ID NO:2.

Moreover, nucleic acid molecules encoding other hydroxylase familymembers and, thus, which have a nucleotide sequence which differs fromthe hydroxylase sequences of SEQ ID NO:1 are intended to be within thescope of the invention. For example, another hydroxylase cDNA can beidentified based on the nucleotide sequence of SEQ ID NO:1. Moreover,nucleic acid molecules encoding hydroxylase proteins from differentspecies, and which, thus, have a nucleotide sequence which differs fromthe hydroxylase sequences of SEQ ID NO:1, are intended to be within thescope of the invention. For example, Schizochytrium or Crythecodiniumhydroxylase cDNA can be identified based on the nucleotide sequence of aC. purpurea fatty acid hydroxylase.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the hydroxylase cDNAs of the invention can be isolatedbased on their homology to the hydroxylase nucleic acids disclosedherein using the cDNAs disclosed herein, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions.

Orthologues, homologues and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1 or acomplement of the nucleotide sequence of SEQ ID NO:1, for example, theexact complement of the nucleotide sequence of SEQ ID NO:1. In otherembodiment, the nucleic acid is at least 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 1000 or 1250 or morenucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or alternativelyhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or alternativelyhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including, but not limited toblocking agents (e.g., BSA or salmon or herring sperm carrier DNA),detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP andthe like. When using nylon membranes, in particular, an additionalpreferred, non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995), oralternatively 0.2×SSC, 1% SDS.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1corresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the hydroxylasesequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, thereby leading to changes in theamino acid sequence of the encoded hydroxylase proteins, withoutaltering the functional ability of the hydroxylase proteins. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of C. purpurea fatty acidhydroxylase (e.g., the sequence of SEQ ID NO:2) without altering thebiological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues thatare conserved between the hydroxylase proteins of the present inventionand other members of the fatty acid hydroxylase family are not likely tobe amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding hydroxylase proteins that contain changes in aminoacid residues that are not essential for activity. Such hydroxylaseproteins differ in amino acid sequence from SEQ ID NO:2, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably atleast about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%,and even more preferably at least about 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:2, e.g., to the entire length of SEQ ID NO:2.

An isolated nucleic acid molecule encoding a hydroxylase proteinhomologous to the protein of SEQ ID NO:2 can be created by introducingone or more nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO:1 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a hydroxylase protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a hydroxylase coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for hydroxylase biological activity to identify mutants thatretain activity. Following mutagenesis of SEQ ID NO:1, the encodedprotein can be expressed recombinantly and the activity of the proteincan be determined.

In a preferred embodiment, a mutant hydroxylase protein can be assayedfor the ability to (i) interact with a hydroxylase substrate or targetmolecule (e.g., a fatty acid) and/or (ii) form a hydroxyl group in ahydroxylase substrate or target molecule using standard assays known inthe art or those assays described herein, for example, in the Examples.

II. Isolated Hydroxylase Proteins

One aspect of the invention pertains to isolated or recombinanthydroxylase proteins and polypeptides, and biologically active portionsthereof. In one embodiment, native hydroxylase proteins can be isolatedfrom cells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment,hydroxylase proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a hydroxylase protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thehydroxylase protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofhydroxylase protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of hydroxylase protein havingless than about 80%, 70%, 60%, 50%, 40%, or 30% (by dry weight) ofnon-hydroxylase protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-hydroxylaseprotein, still more preferably less than about 10% of non-hydroxylaseprotein, and most preferably less than about 5% non-hydroxylase protein.When the hydroxylase protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of hydroxylase protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of hydroxylase protein having less than about 30%(by dry weight) of chemical precursors or non-hydroxylase chemicals,more preferably less than about 20% chemical precursors ornon-hydroxylase chemicals, still more preferably less than about 10%chemical precursors or non-hydroxylase chemicals, and most preferablyless than about 5% chemical precursors or non-hydroxylase chemicals. Itshould be understood that the proteins of this invention can also be ina form which is different than their corresponding naturally occurringproteins and/or which is still in association with at least somecellular components. For example, the protein can be associated with acellular membrane.

As used herein, a “biologically active portion” of a hydroxylase proteinincludes a fragment of a hydroxylase protein which participates in aninteraction between a hydroxylase molecule and a non-hydroxylasemolecule (e.g., a hydroxylase substrate such as fatty acid).Biologically active portions of a hydroxylase protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the hydroxylase amino acid sequences, e.g., the amino acidsequences shown in SEQ ID NO:2 which include sufficient amino acidresidues to exhibit at least one activity of a hydroxylase protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the hydroxylase protein, for example, theability to (i) interact with a hydroxylase substrate or target molecule(e.g., a fatty acid) and/or (ii) form a hydroxyl group in a hydroxylasesubstrate or target molecule, A biologically active portion of ahydroxylase protein can be a polypeptide which is, for example, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400 or 450 or more amino acids in length.

In one embodiment, a biologically active portion of a hydroxylaseprotein comprises a domain conserved among hydroxylases and known toparticipate in a hydroxylase activity. For example, at least one domainor motif conserved among at least two, at least three or the four aminoacid sequences encoding fatty acid hydroxylases from differentorganisms, as depicted in FIG. 2, can be incorporated within thebiologically active fragments in order to preserve hydroxylase activity.Specifically, hydroxylases often possess the following conserved aminoacid domains: GHECGH (SEQ ID NO: 6), HSAHH (SEQ ID NO: 7) and HVVHH (SEQID NO: 8). Accordingly, in particular embodiments of the presentinvention, biologically active fragments of polypeptides include atleast one domain selected from the group consisting of GHECGH (SEQ IDNO: 6), HSAHH (SEQ ID NO: 7) and HVVHH (SEQ ID NO: 8). In otherembodiments, nucleic acid molecules encoding for biologically activefragments include nucleotide sequences encoding for at least one domainselected from the group consisting of GHECGH (SEQ ID NO: 6), HSAHH (SEQID NO: 7) and HVVHH (SEQ ID NO: 8). Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native hydroxylase protein.

In a preferred embodiment, a hydroxylase protein has an amino acidsequence shown in SEQ ID NO:2. In other embodiments, the hydroxylaseprotein is substantially identical to SEQ ID NO:2 and retains thefunctional activity of the protein of SEQ ID NO:2, yet differs in aminoacid sequence due to natural allelic variation or mutagenesis, asdescribed in detail in subsection I above. In another embodiment, thehydroxylase protein is a protein which comprises an amino acid sequenceat least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%,preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or90%, 91%, 92%, 93%, 94%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.

In another embodiment, the invention features a hydroxylase proteinwhich is encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, or 90%, 91%, 92%, 93%, 94%, and even more preferably at leastabout 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequenceof SEQ ID NO:1, or a complement thereof. This invention further featuresa hydroxylase protein which is encoded by a nucleic acid moleculeconsisting of a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the C. purpurea fattyacid hydroxylase amino acid sequence of SEQ ID NO:2 having 477 aminoacid residues, at least 143, preferably at least 191, more preferably atleast 238, even more preferably at least 286, and even more preferablyat least 334, 382, or 429 amino acid residues are aligned). The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available atwww.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and agap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package (available at www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting exampleof parameters to be used in conjunction with the GAP program include aBlosum 62 scoring matrix with a gap penalty of 12, a gap extend penaltyof 4, and a frameshift gap penalty of 5.

In another embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of Meyers andMiller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM 120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to hydroxylase nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to hydroxylaseprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seewww.ncbi.nlm.nih.gov.

III. Methods of Producing Hydroxyl Fatty Acids

The present invention provides new and improved methods of producinghydroxyl fatty acids, e.g., 12-hydroxyoctadec-9-enoic acid (ricinoleicacid) or lesqueroileic acid.

A. Recombinant Cells and Methods for Culturing Cells

The present invention further features recombinant vectors that includenucleic acid sequences that encode the gene products as describedherein, preferably hydroxylase gene products. The term recombinantvector includes a vector (e.g., plasmid) that has been altered, modifiedor engineered such that it contains greater, fewer or different nucleicacid sequences than those included in the native vector or plasmid. Inone embodiment, a recombinant vector includes the nucleic acid sequenceencoding at least one fatty acid hydroxylase enzyme operably linked toregulatory sequences. The phrase “operably linked to regulatorysequence(s)” means that the nucleotide sequence of interest is linked tothe regulatory sequence(s) in a manner which allows for expression(e.g., enhanced, increased, constitutive, basal, attenuated, decreasedor repressed expression) of the nucleotide sequence, preferablyexpression of a gene product encoded by the nucleotide sequence (e.g.,when the recombinant vector is introduced into a cell). Exemplaryvectors are described in further detail herein as well as in, forexample, Frascotti et al., U.S. Pat. No. 5,721,137, the contents ofwhich are incorporated herein by reference.

The term “regulatory sequence” includes nucleic acid sequences whichaffect (e.g., modulate or regulate) expression of other (non-regulatory)nucleic acid sequences. In one embodiment, a regulatory sequence isincluded in a recombinant vector in a similar or identical positionand/or orientation relative to a particular gene of interest as isobserved for the regulatory sequence and gene of interest as it appearsin nature, e.g., in a native position and/or orientation. For example, agene of interest (e.g., a C. purpurea fatty acid hydroxylase gene) canbe included in a recombinant vector operably linked to a regulatorysequence which accompanies or is adjacent to the gene in the naturalorganism (e.g., operably linked to “native” fatty acid regulatorysequence such as the “native” fatty acid hydroxylase promoter).Alternatively, a gene of interest (e.g., a fatty acid hydroxylase gene)can be included in a recombinant vector operably linked to a regulatorysequence which accompanies or is adjacent to another (e.g., a different)gene in the natural organism. For example, a fatty acid hydroxylase genecan be included in a vector operably linked to non-fatty acidhydroxylase regulatory sequences. Alternatively, a gene of interest(e.g., a fatty acid hydroxylase gene) can be included in a vectoroperably linked to a regulatory sequence from another organism. Forexample, regulatory sequences from other microbes (e.g., other bacterialregulatory sequences, bacteriophage regulatory sequences and the like)can be operably linked to a particular gene of interest.

Preferred regulatory sequences include promoters, enhancers, terminationsignals and other expression control elements (e.g., binding sites fortranscriptional and/or translational regulatory proteins, for example,in the transcribed mRNA). Such regulatory sequences are described, forexample, in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in a cell (e.g., constitutive promoters andstrong constitutive promoters), those which direct inducible expressionof a nucleotide sequence in a cell (e.g., inducible promoters, forexample, xylose inducible promoters) and those which attenuate orrepress expression of a nucleotide sequence in a cell (e.g., attenuationsignals or repressor sequences). It is also within the scope of thepresent invention to regulate expression of a gene of interest byremoving or deleting regulatory sequences. For example, sequencesinvolved in the negative regulation of transcription can be removed suchthat expression of a gene of interest is enhanced.

In one embodiment, a recombinant vector of the present inventionincludes nucleic acid sequences that encode at least one gene product(e.g., C. purpurea fatty acid hydroxylase) operably linked to a promoteror promoter sequence.

In a particular embodiment, seed-specific promoters are utilized toenhance the production of the desired hydroxyl fatty acid. For example,U.S. Patent Publication No. 2003-0159174, published Aug. 21, 2003, theentire contents of which are hereby expressly incorporated by referenceherein, describes the use of particular seed-specific promotersincluding, for example, Conlinin 1, Conlinin 2 and LuFad3 from the genusLinum. One skilled in the art will appreciate that other promoters, forexample, seed specific promoters such as napin, may be utilized tomodulate, for example, enhance, the expression of the hydroxylasenucleotide sequence.

In yet another embodiment, a recombinant vector of the present inventionincludes a terminator sequence or terminator sequences (e.g.,transcription terminator sequences). The term “terminator sequences”includes regulatory sequences which serve to terminate transcription ofmRNA. Terminator sequences (or tandem transcription terminators) canfurther serve to stabilize mRNA (e.g., by adding structure to mRNA), forexample, against nucleases.

In yet another embodiment, a recombinant vector of the present inventionincludes antibiotic resistance sequences. The term “antibioticresistance sequences” includes sequences which promote or conferresistance to antibiotics on the host organism. In one embodiment, theantibiotic resistance sequences are selected from the group consistingof cat (chloramphenicol resistance), tet (tetracycline resistance)sequences, erm (erythromycin resistance) sequences, neo (neomycinresistance) sequences and spec (spectinomycin resistance) sequences.Recombinant vectors of the present invention can further includehomologous recombination sequences (e.g., sequences designed to allowrecombination of the gene of interest into the chromosome of the hostorganism). For example, amyE sequences can be used as homology targetsfor recombination into the host chromosome.

The term “manipulated cell” includes a cell that has been engineered(e.g., genetically engineered) or modified such that the cell has atleast one fatty acid hydroxylase of the invention (e.g., SEQ ID NO:1),such that a hydroxyl fatty acid is produced. Modification or engineeringof such microorganisms can be according to any methodology describedherein including, but not limited to, deregulation of a biosyntheticpathway and/or overexpression of at least one biosynthetic enzyme. A“manipulated” enzyme (e.g., a “manipulated” biosynthetic enzyme)includes an enzyme, the expression or production of which has beenaltered or modified such that at least one upstream or downstreamprecursor, substrate or product of the enzyme is altered or modified,for example, as compared to a corresponding wild-type or naturallyoccurring enzyme.

The term “overexpressed” or “overexpression” includes expression of agene product (e.g., a fatty acid hydroxylase) at a level greater thanthat expressed prior to manipulation of the cell or in a comparable cellwhich has not been manipulated. In one embodiment, the cell can begenetically manipulated (e.g., genetically engineered) to overexpress alevel of gene product greater than that expressed prior to manipulationof the cell or in a comparable cell which has not been manipulated.Genetic manipulation can include, but is not limited to, altering ormodifying regulatory sequences or sites associated with expression of aparticular gene (e.g., by adding strong promoters, inducible promotersor multiple promoters or by removing regulatory sequences such thatexpression is constitutive), modifying the chromosomal location of aparticular gene, altering nucleic acid sequences adjacent to aparticular gene such as a ribosome binding site or transcriptionterminator, increasing the copy number of a particular gene, modifyingproteins (e.g., regulatory proteins, suppressors, enhancers,transcriptional activators and the like) involved in transcription of aparticular gene and/or translation of a particular gene product, or anyother conventional means of deregulating expression of a particular generoutine in the art (including but not limited to use of antisensenucleic acid molecules, for example, to block expression of repressorproteins).

In another embodiment, the cell can be physically or environmentallymanipulated to overexpress a level of gene product greater than thatexpressed prior to manipulation of the cell or in a comparable cellwhich has not been manipulated. For example, a cell can be treated withor cultured in the presence of an agent known or suspected to increasetranscription of a particular gene and/or translation of a particulargene product such that transcription and/or translation are enhanced orincreased. Alternatively, a cell can be cultured at a temperatureselected to increase transcription of a particular gene and/ortranslation of a particular gene product such that transcription and/ortranslation are enhanced or increased.

The term “deregulated” or “deregulation” includes the alteration ormodification of at least one gene in a cell that encodes an enzyme in abiosynthetic pathway, such that the level or activity of thebiosynthetic enzyme in the cell is altered or modified. Preferably, atleast one gene that encodes an enzyme in a biosynthetic pathway isaltered or modified such that the gene product is enhanced or increased.The phrase “deregulated pathway” can also include a biosynthetic pathwayin which more than one gene that encodes an enzyme in a biosyntheticpathway is altered or modified such that the level or activity of morethan one biosynthetic enzyme is altered or modified. The ability to“deregulate” a pathway (e.g., to simultaneously deregulate more than onegene in a given biosynthetic pathway) in a cell arises from theparticular phenomenon of cells in which more than one enzyme (e.g., twoor three biosynthetic enzymes) are encoded by genes occurring adjacentto one another on a contiguous piece of genetic material termed an“operon”.

The term “operon” includes a coordinated unit of gene expression thatcontains a promoter and possibly a regulatory element associated withone or more, preferably at least two, structural genes (e.g., genesencoding enzymes, for example, biosynthetic enzymes). Expression of thestructural genes can be coordinately regulated, for example, byregulatory proteins binding to the regulatory element or byanti-termination of transcription. The structural genes can betranscribed to give a single mRNA that encodes all of the structuralproteins. Due to the coordinated regulation of genes included in anoperon, alteration or modification of the single promoter and/orregulatory element can result in alteration or modification of each geneproduct encoded by the operon. Alteration or modification of theregulatory element can include, but is not limited to removing theendogenous promoter and/or regulatory element(s), adding strongpromoters, inducible promoters or multiple promoters or removingregulatory sequences such that expression of the gene products ismodified, modifying the chromosomal location of the operon, alteringnucleic acid sequences adjacent to the operon or within the operon suchas a ribosome binding site, increasing the copy number of the operon,modifying proteins (e.g., regulatory proteins, suppressors, enhancers,transcriptional activators and the like) involved in transcription ofthe operon and/or translation of the gene products of the operon, or anyother conventional means of deregulating expression of genes routine inthe art (including but not limited to use of antisense nucleic acidmolecules, for example, to block expression of repressor proteins).Deregulation can also involve altering the coding region of one or moregenes to yield, for example, an enzyme that is feedback resistant or hasa higher or lower specific activity.

A particularly preferred “recombinant” cell of the present invention hasbeen genetically engineered to overexpress a plant-derived gene or geneproduct or an microorganismally-derived gene or gene product. The term“plant-derived,” “microorganismally-derived,” or “derived-from,” forexample, includes a gene which is naturally found in a microorganism ora plant, e.g., an oilseed plant, or a gene product (e.g., the fatty acidhydroxylase of SEQ ID NO:2) or which is encoded by a plant gene or agene from a microorganism (e.g., encoded SEQ ID NO:1).

The methodologies of the present invention feature recombinant cellswhich overexpress at least one fatty acid hydroxylase. In oneembodiment, a recombinant cell of the present invention has beengenetically engineered to overexpress a Claviceps fatty acid hydroxylase(e.g., a fatty acid hydroxylase having the amino acid sequence of SEQ IDNO:2 or encoded by the nucleic acid sequence of SEQ ID NO:1).

In another embodiment, the invention features a cell (e.g., a plant ormicrobial cell) that has been transformed with a vector comprising afatty acid hydroxylase nucleic acid sequence (e.g., a fatty acidhydroxylase nucleic acid sequence as set forth in SEQ ID NO:1).

Another aspect of the present invention features a method of modulatingthe production of hydroxyl fatty acids comprising culturing cellstransformed by the nucleic acid molecules of the present invention(e.g., a hydroxylase) such that modulation of hydroxyl fatty acidproduction occurs (e.g., production of hydroxyl fatty acids isenhanced). The method of culturing cells transformed by the nucleic acidmolecules of the present invention to modulate the production of fattyacids is referred to herein as “biotransformation.” Thebiotransformation processes can utilize recombinant cells and/orhydroxylases described herein. The term “biotransformation process,”also referred to herein as “bioconversion processes,” includesbiological processes which result in the production (e.g.,transformation or conversion) of any compound (e.g., substrate,intermediate, or product) which is upstream of a fatty acid hydroxylaseto a compound (e.g., substrate, intermediate, or product) which isdownstream of a fatty acid hydroxylase, in particular, a hydroxyl fattyacid. In one embodiment, the invention features a biotransformationprocess for the production of a hydroxyl fatty acid comprisingcontacting a cell which overexpresses at least one fatty acidhydroxylase with at least one appropriate substrate, for example, anunhydroxylated fatty acid, under conditions such that a hydroxyl fattyacid is produced and, optionally, recovering the fatty acid. In apreferred embodiment, the invention features a biotransformation processfor the production of hydroxyl fatty acids comprising contacting a cellwhich overexpresses a fatty acid hydroxylase with an appropriatesubstrate (e.g., an intermediate fatty acid) under conditions such thata hydroxyl fatty acid (e.g., ricinoleic and lesqueroleic acid) isproduced and, optionally, recovering the hydroxyl fatty acid. Conditionsunder which a hydroxyl fatty acid is produced can include any conditionswhich result in the desired production of a hydroxyl fatty acid.

The cell(s) and/or enzymes used in the biotransformation reactions arein a form allowing them to perform their intended function (e.g.,producing a desired hydroxyl fatty acid). The cells can be whole cells,or can be only those portions of the cells necessary to obtain thedesired end result. The cells can be suspended (e.g., in an appropriatesolution such as buffered solutions or media), rinsed (e.g., rinsed freeof media from culturing the cell), acetone-dried, immobilized (e.g.,with polyacrylamide gel or k-carrageenan or on synthetic supports, forexample, beads, matrices and the like), fixed, cross-linked orpermeablized (e.g., have permeablized membranes and/or walls such thatcompounds, for example, substrates, intermediates or products can moreeasily pass through said membrane or wall).

The type of cell can be any cell capable of being used within themethods of the invention, e.g., plant, animal, or microbial cells,preferably a plant or microbial cell. In one embodiment, the cell is aplant cell, for example, an oilseed plant, including, but not limitedto, flax (Linum sp.), rapeseed (Brassica sp.), soybean (Glycine and Sojasp.), sunflower (Helianthus sp.), cotton (Gossypium sp.), corn (Zeamays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobromacacoa), peanut (Arachis sp.), hemp, camelina, crambe, oil palm,coconuts, groundnuts, sesame seed, castor bean, lesquerella, tallowtree, sheanuts, tungnuts, kapok fruit, poppy seed, jojoba seeds andperilla. In another embodiment, the cell is Brassica juncea. U.S. PatentPublication No. 2003-0159174, published Aug. 21, 2003, the entirecontents of which are hereby expressly incorporated by reference herein,provides extensive teaching on the transformation of plant cells tooptimize production of a desired end product.

In yet another embodiment, the cell is a microbial cell, for example,Candida, Cryptococcus, Lipomyces, Rhodosporidium, Yarrowia,Thraustochytrium, Pythium irregulare, Schizochytrium and Cythecodinium.One skilled in the art will appreciate that other microbial cells can beused in accordance with the methods provided herein, for example, forthe production of a hydroxyl fatty acid.

An important aspect of the present invention involves growing therecombinant plant or culturing the recombinant microorganisms describedherein, such that a desired compound (e.g., a desired hydroxyl fattyacid) is produced. The term “culturing” includes maintaining and/orgrowing a living microorganism of the present invention (e.g.,maintaining and/or growing a culture or strain). In one embodiment, amicroorganism of the invention is cultured in liquid media. In anotherembodiment, a microorganism of the invention is cultured in solid mediaor semi-solid media. In a preferred embodiment, a microorganism of theinvention is cultured in media (e.g., a sterile, liquid media)comprising nutrients essential or beneficial to the maintenance and/orgrowth of the microorganism (e.g., carbon sources or carbon substrate,for example complex carbohydrates such as bean or grain meal, starches,sugars, sugar alcohols, hydrocarbons, oils, fats, fatty acids, organicacids and alcohols; nitrogen sources, for example, vegetable proteins,peptones, peptides and amino acids derived from grains, beans andtubers, proteins, peptides and amino acids derived form animal sourcessuch as meat, milk and animal byproducts such as peptones, meat extractsand casein hydrolysates; inorganic nitrogen sources such as urea,ammonium sulfate, ammonium chloride, ammonium nitrate and ammoniumphosphate; phosphorus sources, for example, phosphoric acid, sodium andpotassium salts thereof; trace elements, for example, magnesium, iron,manganese, calcium, copper, zinc, boron, molybdenum, and/or cobaltsalts; as well as growth factors such as amino acids, vitamins, growthpromoters and the like).

Preferably, microorganisms of the present invention are cultured undercontrolled pH. The term “controlled pH” includes any pH which results inproduction of the desired product (e.g., a hydroxyl fatty acid). In oneembodiment, microorganisms are cultured at a pH of about 7. In anotherembodiment, microorganisms are cultured at a pH of between 6.0 and 8.5.The desired pH may be maintained by any number of methods known to thoseskilled in the art.

Also preferably, microorganisms of the present invention are culturedunder controlled aeration. The term “controlled aeration” includessufficient aeration (e.g., oxygen) to result in production of thedesired product (e.g., a hydroxyl fatty acid). In one embodiment,aeration is controlled by regulating oxygen levels in the culture, forexample, by regulating the amount of oxygen dissolved in culture media.Preferably, aeration of the culture is controlled by agitating theculture. Agitation may be provided by a propeller or similar mechanicalagitation equipment, by revolving or shaking the growth vessel (e.g.,fermentor) or by various pumping equipment. Aeration may be furthercontrolled by the passage of sterile air or oxygen through the medium(e.g., through the fermentation mixture). Also preferably,microorganisms of the present invention are cultured without excessfoaming (e.g., via addition of antifoaming agents).

Moreover, plants or microorganisms of the present invention can becultured under controlled temperatures. The term “controlledtemperature” includes any temperature which results in production of thedesired product (e.g., a hydroxyl fatty acid). In one embodiment,controlled temperatures include temperatures between 15° C. and 95° C.In another embodiment, controlled temperatures include temperaturesbetween 15° C. and 70° C. Preferred temperatures are between 20° C. and55° C., more preferably between 30° C. and 45° C. or between 30° C. and50° C.

Microorganisms can be cultured (e.g., maintained and/or grown) in liquidmedia and preferably are cultured, either continuously orintermittently, by conventional culturing methods such as standingculture, test tube culture, shaking culture (e.g., rotary shakingculture, shake flask culture, etc.), aeration spinner culture, orfermentation. In a preferred embodiment, the microorganisms are culturedin shake flasks. In a more preferred embodiment, the microorganisms arecultured in a fermentor (e.g., a fermentation process). Fermentationprocesses of the present invention include, but are not limited to,batch, fed-batch and continuous methods of fermentation. The phrase“batch process” or “batch fermentation” refers to a closed system inwhich the composition of media, nutrients, supplemental additives andthe like is set at the beginning of the fermentation and not subject toalteration during the fermentation, however, attempts may be made tocontrol such factors as pH and oxygen concentration to prevent excessmedia acidification and/or microorganism death. The phrase “fed-batchprocess” or “fed-batch” fermentation refers to a batch fermentation withthe exception that one or more substrates or supplements are added(e.g., added in increments or continuously) as the fermentationprogresses. The phrase “continuous process” or “continuous fermentation”refers to a system in which a defined fermentation media is addedcontinuously to a fermentor and an equal amount of used or “conditioned”media is simultaneously removed, preferably for recovery of the desiredproduct (e.g., a hydroxyl fatty acid). A variety of such processes havebeen developed and are well-known in the art.

The phrase “culturing under conditions such that a desired compound isproduced” includes maintaining and/or growing plants or microorganismsunder conditions (e.g., temperature, pressure, pH, duration, etc.)appropriate or sufficient to obtain production of the desired compoundor to obtain desired yields of the particular compound being produced,for example, a hydroxyl fatty acid such as ricinoleic or lesqueroleicacid. For example, culturing is continued for a time sufficient toproduce the desired amount of a hydroxyl fatty acid. Preferably,culturing is continued for a time sufficient to substantially reachmaximal production of the hydroxyl fatty acid. In one embodiment,culturing is continued for about 12 to 24 hours. In another embodiment,culturing is continued for about 24 to 36 hours, 36 to 48 hours, 48 to72 hours, 72 to 96 hours, 96 to 120 hours, 120 to 144 hours, or greaterthan 144 hours. In another embodiment, culturing is continued for a timesufficient to reach production yields of hydroxyl fatty acids, forexample, cells are cultured such that at least about 15 to 20 g/L ofhydroxyl fatty acids are produced, at least about 20 to 25 g/L hydroxylfatty acids are produced, at least about 25 to 30 g/L hydroxyl fattyacids are produced, at least about 30 to 35 g/L hydroxyl fatty acids areproduced, at least about 35 to 40 g/L hydroxyl fatty acids are produced(e.g., at least about 37 g/L hydroxyl fatty acids) or at least about 40to 50 g/L hydroxyl fatty acids are produced. In yet another embodiment,microorganisms are cultured under conditions such that a preferred yieldof hydroxyl fatty acids, for example, a yield within a range set forthabove, is produced in about 24 hours, in about 36 hours, in about 48hours, in about 72 hours, or in about 96 hours.

In producing hydroxyl fatty acids, it may further be desirable toculture cells of the present invention in the presence of supplementalfatty acid biosynthetic substrates. The term “supplemental fatty acidbiosynthetic substrate” includes an agent or compound which, whenbrought into contact with a cell or included in the culture medium of acell, serves to enhance or increase hydroxyl fatty acid biosynthesis.Supplemental fatty acid biosynthetic substrates of the present inventioncan be added in the form of a concentrated solution or suspension (e.g.,in a suitable solvent such as water or buffer) or in the form of a solid(e.g., in the form of a powder). Moreover, supplemental fatty acidbiosynthetic substrates of the present invention can be added as asingle aliquot, continuously or intermittently over a given period oftime.

The methodology of the present invention can further include a step ofrecovering a desired compound (e.g., a hydroxyl fatty acid). The term“recovering” a desired compound includes extracting, harvesting,isolating or purifying the compound from culture media. Recovering thecompound can be performed according to any conventional isolation orpurification methodology known in the art including, but not limited to,treatment with a conventional resin (e.g., anion or cation exchangeresin, non-ionic adsorption resin, etc.), treatment with a conventionaladsorbent (e.g., activated charcoal, silicic acid, silica gel,cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g.,with a conventional solvent such as an alcohol, ethyl acetate, hexaneand the like), dialysis, filtration, concentration, crystallization,recrystallization, pH adjustment, lyophilization and the like. Forexample, a compound can be recovered from culture media by firstremoving the microorganisms from the culture. Media is then passedthrough or over a cation exchange resin to remove unwanted cations andthen through or over an anion exchange resin to remove unwantedinorganic anions and organic acids having stronger acidities than thehydroxyl fatty acid of interest (e.g., ricinoleic acid).

Preferably, a desired compound of the present invention is “extracted,”“isolated” or “purified” such that the resulting preparation issubstantially free of other components (e.g., free of media componentsand/or fermentation byproducts). The language “substantially free ofother components” includes preparations of desired compound in which thecompound is separated (e.g., purified or partially purified) from mediacomponents or fermentation byproducts of the culture from which it isproduced. In one embodiment, the preparation has greater than about 80%(by dry weight) of the desired compound (e.g., less than about 20% ofother media components or fermentation byproducts), more preferablygreater than about 90% of the desired compound (e.g., less than about10% of other media components or fermentation byproducts), still morepreferably greater than about 95% of the desired compound (e.g., lessthan about 5% of other media components or fermentation byproducts), andmost preferably greater than about 98-99% desired compound (e.g., lessthan about 1-2% other media components or fermentation byproducts). Whenthe desired compound is a hydroxyl fatty acid that has been derivatizedto a salt, the compound is preferably further free (e.g., substantiallyfree) of chemical contaminants associated with the formation of thesalt. When the desired compound is a hydroxyl fatty acid that has beenderivatized to an alcohol, the compound is preferably further free(e.g., substantially free) of chemical contaminants associated with theformation of the alcohol.

In an alternative embodiment, the desired hydroxyl fatty acid is notpurified from the plant or microorganism, for example, when the plant ormicroorganism is biologically non-hazardous (e.g., safe). For example,the entire plant or culture (or culture supernatant) can be used as asource of product (e.g., crude product). In one embodiment, the plant orculture (or culture supernatant) supernatant is used withoutmodification. In another embodiment, the plant or culture (or culturesupernatant) is concentrated. In yet another embodiment, the plant orculture (or culture supernatant) is pulverized, dried, or lyophilized.

B. High Yield Production Methodologies

A particularly preferred embodiment of the present invention is a highyield production method for producing hydroxyl fatty acids, e.g.,ricinoleic acid, comprising culturing a manipulated plant ormicroorganism under conditions such that the hydroxyl fatty acid isproduced at a significantly high yield. The phrase “high yieldproduction method,” for example, a high yield production method forproducing a desired compound (e.g., for producing a hydroxyl fatty acid)includes a method that results in production of the desired compound ata level which is elevated or above what is usual for comparableproduction methods. Preferably, a high yield production method resultsin production of the desired compound at a significantly high yield. Thephrase “significantly high yield” includes a level of production oryield which is sufficiently elevated or above what is usual forcomparable production methods, for example, which is elevated to a levelsufficient for commercial production of the desired product (e.g.,production of the product at a commercially feasible cost). In oneembodiment, the invention features a high yield production method ofproducing hydroxyl fatty acids that includes culturing a manipulatedplant or microorganism under conditions such that a hydroxyl fatty acidis produced at a level greater than 2 g/L. In another embodiment, theinvention features a high yield production method of producing hydroxylfatty acids that includes culturing a manipulated plant or microorganismunder conditions such that a hydroxyl fatty acid is produced at a levelgreater than 10 g/L. In another embodiment, the invention features ahigh yield production method of producing hydroxyl fatty acids thatincludes culturing a manipulated plant or microorganism under conditionssuch that a hydroxyl fatty acid is produced at a level greater than 20g/L. In yet another embodiment, the invention features a high yieldproduction method of producing hydroxyl fatty acids that includesculturing a manipulated plant or microorganism under conditions suchthat a hydroxyl fatty acid is produced at a level greater than 30 g/L.In yet another embodiment, the invention features a high yieldproduction method of producing hydroxyl fatty acids that includesculturing a manipulated plant or microorganism under conditions suchthat a hydroxyl fatty acid is produced at a level greater than 40 g/L.

The invention further features a high yield production method forproducing a desired compound (e.g., for producing a hydroxyl fatty acid)that involves culturing a manipulated plant or microorganism underconditions such that a sufficiently elevated level of compound isproduced within a commercially desirable period of time. In an exemplaryembodiment, the invention features a high yield production method ofproducing hydroxyl fatty acids that includes culturing a manipulatedplant or microorganism under conditions such that a hydroxyl fatty acidis produced at a level greater than 15-20 g/L in 36 hours. In anotherembodiment, the invention features a high yield production method ofproducing hydroxyl fatty acids that includes culturing a manipulatedplant or microorganism under conditions such that a hydroxyl fatty acidsproduced at a level greater than 25-30 g/L in 48 hours. In anotherembodiment, the invention features a high yield production method ofproducing hydroxyl fatty acids that includes culturing a manipulatedplant or microorganism under conditions such that a hydroxyl fatty acidsproduced at a level greater than 35-40 g/L in 72 hours, for example,greater that 37 g/L in 72 hours. In another embodiment, the inventionfeatures a high yield production method of producing hydroxyl fattyacids that includes culturing a manipulated plant or microorganism underconditions such that a hydroxyl fatty acid is produced at a levelgreater than 30-40 g/L in 60 hours, for example, greater that 30, 35 or40 g/L in 60 hours. Values and ranges included and/or intermediatewithin the ranges set forth herein are also intended to be within thescope of the present invention. For example, hydroxyl fatty acidproduction at levels of at least 31, 32, 33, 34, 35, 36, 37, 38 and 39g/L in 60 hours are intended to be included within the range of 30-40g/L in 60 hours. In another example, ranges of 30-35 g/L or 35-40 g/Lare intended to be included within the range of 30-40 g/L in 60 hours.Moreover, the skilled artisan will appreciate that culturing amanipulated microorganism to achieve a production level of, for example,“30-40 g/L in 60 hours” includes culturing the microorganism foradditional time periods (e.g., time periods longer than 60 hours),optionally resulting in even higher yields of a hydroxyl fatty acidbeing produced.

IV. Compositions

The hydroxylase nucleic acid molecules, proteins, and fragments thereof,of the invention can be used to produce hydroxyl fatty acids which canbe incorporated into compositions. Compositions of the present inventioninclude, e.g., biolubricants, functional fluids, ink, paints, coatings,nylons, resins, foams and other biopolymers (see Jaworski and Cahoon(2003), the entire contents of which are hereby expressly incorporatedby reference herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES Example 1 Organisms and Culture Conditions

C. purpurea, provided by Dr. Yu Chen, Department of Plant Science,University of Manitoba, was grown at 25° C. for 14 days in medium C(Mantle and Nisbet, 1976). S. cerevisiae strain InvSc1 (Invitrogen,Carlsbad, Calif.) was used as a heterologous host to express the C.purpurea CpFAH hydroxylase. Yeast cells were grown at 28° C. either incomplex medium (YPD) or synthetic minimal medium (SD).

Example 2 Identification and Cloning of CpFAH hydroxylase cDNA

C. purpurea was reported to be capable of producing ricinoleic acid(12OH-18:1-9) in its sclerotia. To elucidate the mechanism underlyingthe biosynthesis of this hydroxyl fatty acid, we undertook a degenerateRT-PCR cloning strategy. We designed two degenerate primers that weretargeted to conserved regions of Δ¹² desaturases from fungi. By usingthis approach, we identified several Δ¹² desaturase-like genes from thericinoleate-producing tissues.

For reverse transcription-polymerase chain reaction (RT-PCR)experiments, the single stranded cDNA was synthesized by Superscript IIIreverse transcriptase (Invitrogen, Carlsbad, Calif.) using total RNAsfrom sclerotium-forming mycelia of C. purpurea. The cDNA was then usedas the template for the PCR reaction with two degenerate oligonucleotideprimers (DM34: 5′-GCICAYGARTGYGGICAYSRIGCITT-3′ (SEQ ID NO: 9) and DM36:5′-TAIGTDATIGCI ACIARCCARTGRTKIACCCA-3′ (SEQ ID NO: 10)). These primerswere designed based on the conserved amino acid regions of Δ¹²hydroxylase and related proteins. The forward primer was in the firstconserved histidine box and the reverse primer was outside the histidineboxes corresponding to the amino acid sequences WV(N/H)HWLVAITY (SEQ IDNO: 11). To obtain the entire sequences of the cDNA, the 5′ and 3′regions were amplified separately using the Marathon cDNA AmplificationKit (BD Biosciences Clontech, Mountain View, Calif.) according to themanufacturer's instructions. The complete sequences includinguntranslated region were then amplified using specific primers DM61(5′-CACTAGGGCAACGAATTACTCTGC-3′ (SEQ ID NO: 12)) and DM62 (5′-GGACGCCATCGTTGACTTCC-3′ (SEQ ID NO: 13)) by Pfx DNA polymerase (Invitrogen,Carlsbad, Calif.). The resulting bands were gel-purified, cloned into apCR4-TOPO-TA cloning vector (Invitrogen, Carlsbad, Calif.) andsequenced.

The open reading frame of the gene encodes a protein of 477 amino acidsin length (FIG. 1). Sequence comparison revealed that CpFAH shares 40%and 39% of amino acid identity with oleate hydroxylase from castor beanand Lesquerella fendleri, respectively. Higher homology of CpFAH wasfound to Δ12 desaturases from fungal Gibberella fujukuroi and A.nidulans (68% and 61%, respectively) (FIG. 2).

Example 3 Transformation of S. cerevisiae (Yeast) with CpFAH andSubsequent Culturing of the Transformed Strain

The coding region of the cDNA was amplified by PCR using the Pfx DNApolymerase (Invitrogen, Carlsbad, Calif.) with primers DM63(5′-GCGAATTCGAAATGGCTTCCGCTACTCC-3′ (SEQ ID NO: 14)) and DM64(5′-GCGAATTCCTACTGAGTC TTCATTGAAATGG-3′ (SEQ ID NO: 15)) and cloneddirectly into pYES2.1 Topo-TA expression vector (Invitrogen, Carlsbad,Calif.) after Taq DNA polymerase treatment. The sequence of the insertwas confirmed to be identical to the original cDNA and in the senseorientation relative to the GAL1 promoter.

S. cerevisiae strain InvSc1 was transformed with the construct using theS. C. EasyComp Transformation Kit (Invitrogen, Carlsbad, Calif.) withselection on uracil-deficient medium. For assessing the hydroxylaseactivity, recombinant yeast cells were grown to saturation in 25-mlcultures for 48 h at 28° C. on minimal medium (synthetic dropout)lacking uracil. The cultures were then washed and used to inoculate 25ml of induction medium containing 2% galactose. Cultures were incubatedat 20° C. for 3 days. INVSc1 yeast containing the empty plasmid vectorpYES2.1 was used as a negative control.

Results and Discussion: Expression of CpFAH in Yeast Resulted in theProduction of Ricinoleic Acid

Transformants containing CpFAH produced several novel fatty acidscompared to the control yeast. However, the most abundant fatty acidproduced in the transformants had the retention time identical tostandard ricinoleic acid and accounted for 15% of total fatty acids(FIG. 3). Mass spectrometry of the derivative of this novel peak showedthat it produced an equivalent mass spectrum to that of derivatizedricinoleate (FIG. 4). Three characteristic ions with m/z values 187,270, and 299 correspond to the three major fragmentation ofTMS-methylricinoleate (FIG. 4). Thus, on the basis of chromatographicretention and mass spectrum, the novel fatty acid was unambiguouslyidentified as ricinoleic acid. These data indicate that CpFAH is anoleate hydroxylase from C. purpurea capable of introducing a hydroxylgroup at position 12 of oleic acid. The other two new fatty acidsproduced in transgenic yeast were identified as 16:2-9, 12 and 18:2-9,12. These results indicate CpFAH is a bifunctional enzyme with Δ¹²desaturase and hydroxylase activities and that the activity ofhydroxylase is higher than that of Δ¹² desaturase.

Example 4 Fatty Acid Analysis

For fatty acid analysis, yeast cells were pelleted by centrifugation,washed once with 0.1% tergitol and once with water. The fatty acids wereconverted to their methyl esters with 3 N methanolic HCl at 80° C. for 1hour. After the addition of 1 mL of water, the sample was extractedtwice with 2 mL of hexane. The hexane extract was combined and driedunder N₂, and resuspended in 200 μL of hexane and analyzed on aHewlett-Packard 5890A gas chromatograph equipped with a DB-23 column(30-m×0.25-mm×0.25-μm). The temperature program was isothermal 160° C.for 1 min, gradient 4° C./min to 240° C., and then isothermal at 240° C.for 10 min. For GC/MS analysis of TMS-recinoleate methyl ester, the 200μL of ricinoleic methyl ester were dried under a stream of nitrogen andthe residue was dissolved in 100 μL of N,O-bis(trimethylsilyl)acetamide(BSA; Aldrich)/pyridine (1:1). GC/MS analysis was accomplished using anAgilent 5973 mass selective detector coupled to an Agilent 6890N gaschromatograph using G1701DA MSD Chemstation software (for instrumentcontrol and data analysis) and equipped with a 30-m×0.25-mm DB-23 columnwith 0.25-μm film thickness (J&W Scientific, Folsom, Calif.). Thechromatograph conditions included a split injection (20:1) onto thecolumn using a helium flow of 0.4 ml/min, an initial temperature of 160°C. for 1 min, and a subsequent temperature ramp of 4° C./min to 240° C.The mass selective detector was run under standard electron impactconditions (70 eV), scanning an effective m/z range of 40-700 at 2.26scans/s.

Example 5 Expression of CpFAH in Plants

To produce ricinoleic acid in plants, the CpFAH cDNA was expressed inArabidopsis thaliana under the control of seed-specific Brassica napusnapin storage protein promoter. The binary vectors (FIG. 5) containingthe candidate gene was introduced by the in-plantaAgrobacterium-infiltration approach into an A. thaliana double mutant(fad2fae1) that is unable to synthesize 20:1-11 and 18:2-9, 12 from 18:1-9, and accumulate a high level of oleic acid. By using this approach,16 transgenic plants were produced. Fatty acid analysis of single seedsindicated the C. purpurea hydroxylase is highly active in A. thaliana.As shown in FIG. 6, compared to the untransformed mutant, transgenic A.thaliana produced three new fatty acids, 18:2-9, 12, 12-hydroxyl-18:1-9and 12-hydroxyl-18:2-9, 15. Among them, 12-hydroxyl-18:1-9 is the mostabundant, followed by 18:2-9, 12 and 12-hydroxyl-18:2-9, 15. Theproduction of hydroxyl fatty acids is depicted in Table 1:

TABLE 1 Hydroxyl fatty acid production in Arabidopsis thaliana strainsSum of Fatty acid analysis 18:1- 18:1- 18:1- 18:2- Hydroxy (wt %) 16:018:0 9c 11c 18:2 18:3 20:0 20:1 OH OH FAs fad2fae1-control-1 4.82 3.3782.53 3.12 1.96 2.3 1.14 0.76 0 0 0 fad2fae1-control-2 6.17 4.43 79.673.21 2.35 2.49 1.02 0.65 0 0 0 castor bean 3.36 6.05 60.91 4.19 2.391.57 0.9 0 15.32 5.31 20.63 fad2fae1/4-6 7.36 6.61 44.81 4.42 9.65 3.370 0 17.85 5.93 23.78 fad2fae1/6-4 12.76 10.87 22.58 13.8 14.43 3.97 1.150 19.24 1.19 20.43 fad2fae1/7-5 7 9.61 40.77 4.8 10.16 3.71 1.03 0 17.55.42 22.92 fad2fae1/8-2 12.86 15.54 25 6.25 16.25 4.64 0 0 16.96 2.519.46 fad2fae1/9-7 2.67 9.37 37.9 5.77 11.5 3.93 0 0 23.55 5.27 28.82fad2fae1/10-1 5.78 9.74 39.96 5.38 10.65 3.55 1.12 0 19.37 4.46 23.83fad2fae1/11-4 9.23 7.86 41.16 5.3 11.59 3.73 0 0 16.8 4.32 21.12fad2fae1/13-3 9.26 8.56 41.98 5.25 11.81 3.63 0.85 0 14.74 3.94 18.68fad2fae1/14-5 9.93 7.25 44.9 4.35 9.54 4.05 0.73 0 13.52 5.73 19.25fad2fae1/15-7 3.07 6.34 42.6 4.85 10.44 3.2 0 0 23.2 6.3 29.5fad2fae1/16-3 3.92 6.2 43.17 5.15 9.42 3.94 0 0 19.89 8.31 28.2

The highest level of ricinoleic acid in transgenic A. thaliana accountedfor 23.5% of the total fatty acid in seeds. The total hydroxyl fattyacids (12-hydroxyl-18:1-9 and 12-hydroxyl-18:2-9, 15) reached up toapproximately 30% of the total fatty acid in seeds. These resultsfurther indicate, in part, that CpFAH is a bifunctional enzyme with Δ¹²desaturase and hydroxylase activities and that the activity ofhydroxylase is higher than that of Δ¹² desaturase.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCE LIST

-   Billault, I., Mantle, P. G., and Robins, R. J. (2004). Deuterium NMR    used to indicate a common mechanism for the biosynthesis of    ricinoleic acid by Ricinus communis and Claviceps purpurea. J. Am.    Chem. Soc. 126, 3250-3256.-   Broun, P., Boddupalli, S., and Somerville, C. (1998). A bifunctional    oleate 12-hydroxylase: desaturase from Lesquerella fendleri.    Plant J. 13, 201-210.-   Broun, P. and Somerville, C. (1997). Accumulation of ricinoleic,    lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis    plants that express a fatty acyl hydroxylase cDNA from castor bean.    Plant Physiol 113, 933-942.-   Jaworski, J. and Cahoon, E. B. (2003). Industrial oils from    transgenic plants. Curr. Opin. Plant Biol. 6, 178-184.-   Mantle, P. G. and Nisbet, L. J. (1976). Differentiation of Claviceps    purpurea in axenic culture. J. Gen. Microbiol. 93, 321-334.-   Mey, G., Oeser, B., Lebrun, M. H., and Tudzynski, P. (2002). The    biotrophic, non-appressorium-forming grass pathogen Claviceps    purpurea needs a Fus3/Pmk1 homologous mitogen-activated protein    kinase for colonization of rye ovarian tissue. Mol. Plant. Microbe    Interact. 15, 303-312.-   Morris, L. J., Hall, S. W., and James, A. T. (1966). The    biosynthesis of ricinoleic acid by Claviceps purpurea. Biochem. J.    100, 29C-30C.-   Smith, M. A., Moon, H., Chowrira, G., and Kunst, L. (2003).    Heterologous expression of a fatty acid hydroxylase gene in    developing seeds of Arabidopsis thaliana. Planta 217, 507-516.-   Tudzynski, P., Correia, T., and Keller, U. (2001). Biotechnology and    genetics of ergot alkaloids. Appl. Microbiol. Biotechnol. 57,    593-605.-   van de Loo, F. J., Broun, P., Turner, S., and Somerville, C. (1995).    An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl    desaturase homolog. Proc. Natl. Acad. Sci. U.S.A 92, 6743-6747.

1. An isolated nucleic acid molecule selected from the group consistingof a) an isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1, or the full complement thereof; b) an isolatednucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2, or the full complement thereof; c) anisolated nucleic acid molecule comprising a nucleotide sequence which isat least 85% identical to the entire nucleotide sequence of SEQ ID NO:1,or the full complement thereof; d) an isolated nucleic acid moleculecomprising a nucleotide sequence which hybridizes to the complement ofthe nucleotide sequence of SEQ ID NO:1 in 4× sodium chloride/sodiumcitrate (SSC), at about 65-70° C., followed by one or more washes in1×SSC, at about 65-70° C., or the full complement thereof; e) anisolated nucleic acid molecule comprising a fragment of at least 25contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, or thefull complement thereof; and f) an isolated nucleic acid molecule whichencodes a polypeptide comprising an amino acid sequence which is atleast 85% identical to the entire amino acid sequence of SEQ ID NO:2, orthe full complement thereof.
 2. The isolated nucleic acid molecule ofclaim 1, wherein said nucleic acid molecule encodes a fatty acidhydroxylase protein having an activity of catalyzing the introduction ofa hydroxyl group in a fatty acid.
 3. The isolated nucleic acid moleculeof claim 1, wherein said nucleic acid molecule encodes a fatty aciddesaturase protein having an activity of catalyzing the introduction ofa double bond in a fatty acid.
 4. An isolated nucleic acid moleculecomprising the nucleic acid molecule of claim 1 and a nucleotidesequence encoding a heterologous polypeptide.
 5. A vector comprising thenucleic acid molecule of claim
 1. 6. The vector of claim 5, which is anexpression vector.
 7. An isolated host cell transformed with theexpression vector of claim
 6. 8. The host cell of claim 7, wherein saidcell is a plant cell or a microbial cell.
 9. The host cell of claim 8,wherein said plant cell is a cell obtained from an oilseed crop.
 10. Thehost cell of claim 9, wherein the oilseed crop is selected from thegroup consisting of flax (Linum sp.), rapeseed (Brassica sp.), soybean(Glycine and Soja sp.), sunflower (Helianthus sp.), cotton (Gossypiumsp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.),cocoa (Theobroma cacoa), peanut (Arachis sp.), hemp, camelina, crambe,oil palm, coconuts, groundnuts, sesame seed, castor bean, lesquerella,tallow tree, sheanuts, tungnuts, kapok fruit, poppy seed, jojoba seedsand perilla.
 11. The host cell of claim 8, wherein the microbial cell isselected from the group consisting of Candida, Cryptococcus, Lipomyces,Rhodosporidium, Yarrowia, Thraustochytrium, Pythium, Schizochytrium andCrythecodinium.
 12. The host cell of claim 7, wherein the cell isderived from Saccharomyces cerevisiae.
 13. A method of producing apolypeptide comprising culturing the host cell of claim 7 in anappropriate culture medium to, thereby, produce the polypeptide, whereinthe polypeptide is at least one of a fatty acid hydroxylase proteinhaving an activity of catalyzing the introduction of a hydroxyl group ina fatty acid or a fatty acid desaturase protein having an activity ofcatalyzing the introduction of a double bond in a fatty acid.
 14. Amethod for producing a hydroxyl fatty acid, comprising culturing thecell of claim 7 such that the hydroxyl fatty acid is produced.
 15. Amethod of modulating the production of a hydroxyl fatty acid comprisingculturing the cell of claim 7, such that modulation of the production ofa hydroxyl fatty acid occurs.
 16. The method of claim 15, wherein theproduction of the hydroxyl fatty acid is enhanced.
 17. A method for thelarge-scale production of a hydroxyl fatty acid comprising culturing thecell of claim 7, such that the production of the hydroxyl fatty acidoccurs.
 18. A plant comprising the vector of claim
 6. 19. A seedproduced by the plant of claim
 18. 20. The vector of claim 5, whereinthe nucleic acid molecule is under the control of a seed-specificpromoter.
 21. The vector of claim 20, wherein the seed-specific promoteris selected from the group consisting of Conlinin 1, Conlinin 2, napinand LuFad3 or other seed-specific promoters.
 22. A method of producin ahydroxyl fatty acid comprising contacting a composition comprising atleast one hydroxylase target molecule with at least one polypeptideencoded by the nucleic acid molecule of claim 1 under conditions suchthat the hydroxyl fatty acid is produced.
 23. The method of claim 22,wherein the hydroxylase target molecule is octadec-9-enioc acid (oleicacid).
 24. A method of producing a cell capable of generating a hydroxylfatty acid comprising introducing into said cell the nucleic acidmolecule of claim 1, wherein the nucleic acid molecule encodes ahydroxylase having an activity of catalyzing the introduction of ahydroxyl group in a fatty acid.
 25. The isolated nucleic acid moleculeof claim 1, wherein the nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO:1, or the full complement thereof.
 26. Theisolated nucleic acid molecule of claim 1, wherein the nucleic acidmolecule encodes a polypeptide comprising the amino acid sequence of SEQID NO:2, or the full complement thereof.
 27. The isolated nucleic acidmolecule of claim 1, wherein the nucleic acid molecule comprises anucleotide sequence which is at least 85% identical to the entirenucleotide sequence of SEQ ID NO:1, or the full complement thereof. 28.The nucleic acid molecule of claim 27, wherein the nucleic acid moleculecomprises a nucleotide sequence which is at least 90% identical to theentire nucleotide sequence of SEQ ID NO:1.
 29. The nucleic acid moleculeof claim 27, wherein the nucleic acid molecule comprises a nucleotidesequence which is at least 95% identical to the entire nucleotidesequence of SEQ ID NO:1.
 30. The isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule comprises a nucleotide sequencewhich hybridizes to the complement of the nucleotide sequence of SEQ IDNO:1 in 4X sodium chloride/sodium citrate (SSC), at about 65-70° C.,followed by one or more washes in 1X SSC, at about 65-70° C., or thefull complement thereof.
 31. The isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule comprises a fragment of at least 25contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, or thefull complement thereof.
 32. The isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule encodes a polypeptide comprising anamino acid sequence which is at least 85% identical to the entire aminoacid sequence of SEQ ID NO:2, or the full complement thereof.
 33. Thenucleic acid molecule of claim 32, wherein the polypeptide comprises anamino acid sequence which is at least 90% identical to the entire aminoacid sequence of SEQ ID NO:2.
 34. The nucleic acid molecule of claim 32,wherein the polypeptide comprises an amino acid sequence which is atleast 95% identical to the entire amino acid sequence of SEQ ID NO:2.35. The nucleic acid molecule of claim 1(c), wherein the nucleic acidmolecule comprises a nucleotide sequence which is at least 95% identicalto the entire nucleotide sequence of SEQ ID NO:1.
 36. The nucleic acidmolecule of claim 1(f), wherein the polypeptide comprises an amino acidsequence which is at least 90% identical to the entire amino acidsequence of SEQ ID NO:2.
 37. The nucleic acid molecule of claim 1(f),wherein the polypeptide comprises an amino acid sequence which is atleast 95% identical to the entire amino acid sequence of SEQ ID NO:2.38. The method of claim 37, wherein said plant cell is a cell obtainedfrom an oilseed crop.
 39. The method of claim 38, wherein the oilseedcrop is selected from the group consisting of flax (Linum sp.), rapeseed(Brassica sp.), soybean (Glycine and Soja sp.), sunflower (Helianthussp.), cotton (Gossypium sp.), corn (Zea mays), olive (Olea sp.),safflower (Carthamus sp.), cocoa (Theobroma cacoa), peanut (Arachissp.), hemp, camelina, crambe, oil palm, coconuts, groundnuts, sesameseed, castor bean, lesquerella, tallow tree, sheanuts, tungnuts, kapokfruit, poppy seed, jojoba seeds and perrilla.
 40. The method of claim37, wherein said cell is Brassica juncea.
 41. The method of claim 37,wherein the microbial cell is selected from the group consistingCandida, Cryptococcus, Lipomyces, Rhodosporidium, Yarrowia,Thraustochytrium, Pythium, Schizochytrium and Crythecodinium.
 42. Themethod of any one of claims 14, 24, 15 or 17 wherein said cell isderived from Saccharmoyces cerevisiae.
 43. The method of claim 14, 24,17 wherein expression of the nucleic acid molecule results in modulationof production of said hydroxyl fatty acid.